The research application focuses on understanding the nature of memory T-cells. The hypothesis to be addressed is that precursors of memory helper T-cells represent a unique cellular subset that arises when the naive T-cell first encounters its antigen. The candidate will use a well-defined model system already established in the lab of the mentor. It involves the H2IAk-restricted CD4+ helper cell response to pigeon cyrochrome. This molecule elicits Th cells with a clonally restricted dominant TCR (Vall, Vb3) specific for a defined cytochrome peptide. The CDR3 loop of the TCR is typified by a glutamic acid at position 93 of the a chain and a glutamic acid in position 100 of the b chain. The key to these studies is the ability to follow this dominant T cell receptor. Toward this end, he will use PCR of single cells and a novel biotin-labeled tertrameric molecule containing the Iak protein associated with the defined cytochrome peptide developed by the sponsor and previous mentor, Dr. Mark Davis. Preliminary data and published reports suggest that both approaches are feasible. There are 3 specific aims. The first is to define the microenvironmental distribution of distinct antigen specific populations (presumably reflecting differentiation states). Laser scanning confocal microscopy in combination with the antigen-IAk tetramer will be sued to map the cells within the lymph node architecture and to examine an array of the differentiation antigens. A 7-parameter FACs analysis will be used to more carefully characterize the differentiation phenotype the antigen-specific cells. All of the required serological reagents are available. A possible unanticipated complication is the lack of a track record of the lab in the use of multi-parameter confocal microscopy, the major question being whether the tetameric reagent can be well-visualized. However, even assuming technical complexities, the aim is well conceived and should yield useful data. The second aim is to determine whether the progression to phenotypically distinck antigen-specific T-cell populations is associated with binding kinetics to peptide/MHC class II proteins. One explanation for the transition of memory cells is the selection of T-cells with high affinity for antigen. Using single cell PCR, Dr. Mikszta will determine the TCR usage of cells identified from the preceding studies that display different cell surface phenotypes. She will then clone the TCRs into a plasmid that allows expression of a PI-linked TCR in CHO cells. The TCR can then be retrived in soluble form for binding studies. These studies are made possible by BIAcore analysis that will be carried out in collaboration with Dr. Mark Davis. These studies war technically complex but feasible. The final aim is to analyze the activation requirement for phenotypically distinguishable antigen-specific cell types that are defined in the preceding aims. The FACS-sorted populations will be analyzed in vitro for their cytokine production and proliferaton capability after activation with varous co-ligands and the tetrameric antigen. This is an important extension of the preceding studies, and the candidate recognizes the pitfalls involved in analysis of rare cell types.